Assessing Long‐Term Neurologic Outcomes in SAMD9L ‐Related Ataxia‐Pancytopenia Syndrome

Abstract Background Most published reports on SAMD9L‐related ataxia‐pancytopenia syndrome (ATXPC) have emphasized the hematologic findings. Fewer details are known about the progression of neurologic manifestations and methods for monitoring them. Cases We present six individuals from two families transmitting a heterozygous variant in SAMD9L, exhibiting clinical variations in their hematologic and neurologic findings. Serial motor function testing was used to monitor motor proficiency over a 2 to 3 year period in the proband and his father from Family 1. Conclusions Our case series focuses on the neurologic progression in patients with heterozygous variants in SAMD9L. Patients with ATXPC should be followed to evaluate a wide range of neurologic manifestations. Serial motor function testing using a standardized method is helpful to track changes in balance and coordination in children and adults with ATXPC and could aid in a future extended natural history study.


Introduction
The ataxia-pancytopenia syndrome (ATXPC) was first reported by Li et al 1 in 1978 in a family with variable levels of ataxia and hematologic cytopenias.In 2016, Chen et al2 detected a heterozygous pathogenic variant in SAMD9L in another multigenerational family with ATXPC and a different SAMD9L variant in the family described by Li, confirming SAMD9L as the causative gene.Most published reports on ATXPC focused on familial hematologic disorders, including transient aplastic anemia, myelodysplastic syndrome (MDS) involving monosomy 7, and acute leukemia.More insights into neurologic manifestations and monitoring methods would enhance the understanding of the full clinical spectrum and its pathogenesis.
SAMD9L is a tumor suppressor gene located at chromosome 7q21 that inhibits cell proliferation.Gain-of-function SAMD9L mutations constitutively activate the protein, leading to poor cell division, particularly affecting bone marrow cells.Hematopoietic cells that lose or inactivate the pathogenic variant have a selective advantage.In patients with gain-of-function SAMD9L mutations, the hematopoietic system can undergo somatic reversion through different mechanisms that result in loss or modification of the pathogenic SAMD9L allele.3][4] Patients with monosomy 7/7q have loss of other genes required for normal hematopoiesis, leading to the development of myelodysplasia.In patients with UPD 7q, the part of chromosome 7 carrying the diseasecausing SAMD9L allele is replaced by the wild-type allele bearing homolog, resulting in cells with two normal copies of chromosome 7. 5,6 Patients in whom somatic reversion has occurred through UPD or somatic inactivating variants might have normal hematopoiesis occasionally, but remain at risk for development of CASE SERIES neurologic manifestations. 7These events also explain cases of transient aplastic anemia and disappearance of monosomy 7-myelodysplastic clones. 8Here we report two families with SAMD9L variants clinically classified as pathogenic or as a mosaic variant of uncertain significance (VUS) that demonstrate a range of neurologic and hematologic manifestations, and present data on monitoring of ataxia progression using validated methods in two members of family 1.

Cases Family 1
The proband presented for neurologic evaluation at age 10 years because of progressive predominantly cerebellar ataxia.Family history was significant for two brothers with monosomy 7-MDS.He had normal childhood development.He was found to have mild thrombocytopenia at age 4 years during evaluation as a potential stem cell donor; hematologic work-up was negative for monosomy 7-MDS.Based on his prominent ataxia and family history of monosomy 7-MDS, genetic testing was performed, which revealed a missense VUS in SAMD9L (Table 1), present only in 16% of sequencing-reads, suggestive of mosaicism.The variant was absent from the gnomAD database, and predictive tools indicated the variant likely has a pathologic effect on the protein.At age 11, he was tested on the Bruininks-Oseretsky Test of Motor Proficiency, 2nd edition (BOT-2), 9 and was assessed annually for the period of 3 years (Table 2).The proband's father, age 44-years-old, presented to Neurology at age 19 years for altered gait, dysarthria, and memory loss in the setting of flu-like symptoms.He had been previously healthy with normal development.Three months later, he developed left lower extremity weakness and coordination difficulties with falling, prompting referral to physical therapy.At age 31 years he presented with gradual decline in balance and increased irritability during a viral illness.He reported confusion, word-finding difficulties, and trouble performing simple tasks.Ten years later he was seen for progressive difficulty with walking.At neurology visit 6 months later, the Scale for Assessment and Rating of Ataxia (SARA) 10 was performed and repeated at follow-ups for two years (Table 2).
Under an approved IRB protocol at the University of Washington, Sanger sequencing of blood-derived DNA from the father showed only the wild-type SAMD9L allele.Using allelespecific PCR primers designed to specifically amplify only the variant or the wild type allele, we detected the SAMD9L missense variant seen in his son (Fig. 1), strongly suggesting a low level of mosaicism for cells that retained the germline chromosome 7 bearing the SAM9DL variant.Comparative genomic hybridization (CGH) microarray showed loss-of-heterozygosity of chromosome 7q, consistent with UPD.The sequencing and CGH results suggested that the majority of blood cells were derived from a stem cell that had undergone somatic reversion.
The older of the proband's two younger brothers developed persistent fevers at age 1 year and was diagnosed with monosomy 7-MDS (Table 1).An unrelated allogeneic bone marrow transplant  1).On the BOT-2, he scored below average for age in balance, body coordination and fine motor precision, integration, and control.Both brothers received a presumptive diagnosis of ATXPC, given their family history and the monosomy 7 preceding transplant.

Family 2
The proband presented with pancytopenia and aplastic anemia at age 1 year and had findings of predominantly cerebellar ataxia on neurologic evaluation (age 5 years) and MRI abnormalities (Table 1).He was identified as having a paternally inherited missense variant in SAM9DL, previously reported as a pathogenic variant. 5,11His father had a normal CBC and was reportedly without neurologic symptoms, although a neurologic exam could not be performed.

Discussion
Here we present six individuals from two families identified to have a heterozygous variant in the SAMD9L gene and various hematologic and neurologic findings.5,12 Hematologic abnormalities include pancytopenia, myelodysplasia, often with monosomy 7, or acute myelogenous leukemia.Neurological abnormalities include ataxia (predominantly cerebellar), dysarthria, nystagmus, hyperreflexia, memory and cognitive changes, and white matter changes and cerebellar atrophy on brain MRI. 3,5,12As seen in our families, patients can present with a wide variety of hematologic findings, neurologic findings, or both.
Our case series provides greater detail on the neurologic deficits seen in patients with SAMD9L variants over time.We also demonstrate the value of following ataxia over a period of years using standardized motor function scales (eg, BOT-2 or SARA).For the proband in Family 1, the most severe BOT-2 scores were for balance and remained stable over a period of 3 years.The greatest areas of decline on BOT-2 were in the composites of fine motor integration and bilateral coordination.Fine motor precision and manual dexterity, although below average at initial testing, remained stable.Improvement in upper limb coordination was seen for unclear reasons.Family 1's proband's father had abrupt worsening of SARA scores following a severe motor vehicle collision with prolonged hospitalization and subsequent loss of independent ambulation.Sitting, finger chase, nose-finger, fast alternating hand movements, and heel-shin slide showed only mild worsening over this period, and dysarthria remained stable.Family 1's proband and his father had EMG evidence of sensory axonal polyneuropathy.Sensory or motor peripheral neuropathy has previously been reported in cases of ATXPC. 6The literature suggests that 75% of patients with ATXPC will develop neurologic manifestations and 80% will develop hematologic abnormalities. 3 Patients with pathogenic SAMD9L variants who have not developed or have milder hematologic abnormalities may have had somatic genetic rescue effect in their hematopoietic system through UPD 7q or an inactivating mutation in cis with the aberrant germline SAMD9L allele, a phenomenon that appears to occur frequently in patients with SAMD9L pathogenic variants. 13The time of the somatic reversion occurrence during early development and the repopulation of a tissue could impact revertant clonal clusters size and distribution, which might result in the modification of phenotypic expression in ATXPC cases. 11SAMD9L is currently included on next-generation sequencing panels for ataxia and bone marrow failure.However, further genetic testing needs to be considered in patients with findings consistent with ATXPC in whom no SAMD9L variants are identified on panel testing, given that somatic genetic reversion can eliminate the SAMD9L variant in hematopoietic cells.Evaluation with SNP array, testing using deep target sequencing, and using DNA obtained from other tissues (eg, skin fibroblasts) may be necessary to make a diagnosis. 3,7iven the wide presentation of neurologic symptoms seen in patients with SAMD9L variants and risk for hematologic abnormalities, patients identified as having pathogenic SAMD9L variants should be monitored for both hematologic and neurologic manifestations.Regular testing of coordination and balance using scales such as the BOT-2 or SARA may be helpful to track patients' symptoms over time.Ultimately, a comprehensive scale specific to ATXPC incorporating components from the BOT-2 (in children) or SARA (in adults) would aid in tracking patients over time both individually and collectively as part of a natural history study.This becomes more relevant should prospective therapies emerge, as a disease-specific scale would be needed to assess objective improvement from any therapeutic intervention.

TABLE 1 Continued
(BMT) was performed at age 15 months.At age 8 years, he had no neurologic manifestations.The youngest brother developed fever, rash, and pancytopenia at age 6 months and was diagnosed with monosomy 7-MDS.He received an unrelated allogeneic HLAmatched donor BMT at age 7 months.He had normal childhood development and no neurologic complaints but had subtle abnormalities on neurologic exam at age 6 years (Table

TABLE 2
Ataxia testing for Family 1 proband and proband's father